The specific aims are: A. To develop novel methods using laser desorption extended-range mass spectrometry to qualitatively (and subsequently, semi-quantitatively or quantitatively) determine the presence of multiple neuropeptides derived from a single gene product (e.g. prodynorphin and processed dynorphin A peptides; proenkephalin and processed enkephalin peptides; proopiomelanocortin (POMC) parent peptide and multiple peptides derived from POMC, adrenocorticotropin hormone (ACTH), ?-endorphin, ?-melanocyte-stimulating hormone (MSH) and ?-MSH). B. To use these novel techniques (alone or in combination with HPLC and/or RIA) for the simultaneous qualitative and/or quantitative analysis of opioid peptides and other neuropeptides related to the endogenous opioid system (e.g. Corticotropin releasing factor (CRF), cholecystokinin (CCK), substance P and their processed peptides). C. To use these techniques in studies of the effects of alterations in levels of activity of neurotransmitters on the processing of endogenous opioids (e.g. the effects of altered levels of dopamine on dynorphin processing). D. To use these techniques to study the effects of drugs of abuse and potential treatment agents on opioid peptide processing and degradation (e.g. cocaine, morphine and ethanol effects; methadone, nalmefene and fluoxetine effects). Papers describing this work were published (see below) or are in press. J. Yu, B.T. Chait, L.Toll and M.J. Kreek "Noniceptin in vitro Biotransformation in Human Blood" Peptides 17 (1996) 873-876. 1.5 J. Yu, E.R. Butelman, J.H Woods, B.T. Chait, M. J. Kreek "In vitro Biotransformation of Dynorphin A (1-17) is Similar in Human and Rhesus Monkey Blood as studied by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry" J. Pharmacology and Experimental Therapeutics, 279 (1996) 507 - 514. J. Z. Chou, B.T. Chait, R.Wang, M.J. Kreek "Differential Biotransformation of Dynorphin A (1-17) and Dynorphin A (1-13) Peptides in Human Blood, ex vivo". Peptides. (Tarrytown, NY) 17 (1996) 983-990. J. Yu, E.R. Butelman, J.H. Woods, B.T. Chait, M.J. Kreek "Dynorphin A(1-8) Analog, E-2078, is stable in Human and Rhesus Monkey Blood" J. Pharm. Experimental Therapeutics 280 (1997) 1147-1151.